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A new species and a new record of Helicomyces from Taiwan
Chang-Hsin Kuo
*
, Teik-Khiang Goh
Department of Plant Medicine, National Chiayi University, 300 Syuefu Road, Chiayi City 60004, Taiwan, ROC
article info
Article history:
Received 29 November 2017
Received in revised form
14 March 2018
Accepted 10 April 2018
Available online 26 May 2018
Keywords:
Aquatic hyphomycetes
Freshwater fungi
Phylogenetics
Taxonomy
Tubeufiaceae
abstract
Helicomyces geniculatus sp. nov. from decaying wood submerged in a freshwater stream at Juchi Town of
Alishan area, Chiayi County, Taiwan, is described and illustrated. It differs from other Helicomyces species
in having geniculate conidiophores arising from repent hyphae and also borne on erect setae. Abundant
stalked sclerotia were also found in this species. Helicomyces torquatus is described in this paper as a new
record for Taiwan, with new observation. The phylogenetic relationship of H. geniculatus,H. torquatus,
and related taxa were sought by comparing the sequences of their ITS barcode of the nuc rDNA. A
synopsis of the 13 accepted Helicomyces species is given.
©2018 Published by Elsevier B.V. on behalf of the Mycological Society of Japan.
1. Introduction
Helicosporous fungi have been the subject of systematic studies
because they are morphologically diverse and produce unusual but
elegant conidia for reproduction (Goos, 1987). They are mostly
saprobes on plant litter, rotten wood, decaying twigs in moist pla-
ces or around water (Tsui, Baschien, &Goh, 2016;Wong et al.,
1998). Helicoma Corda, Helicomyces Link, and Helicosporium Nees
are the three earliest erected helicosporous genera. They are ana-
morphs of ascomycetes, and their teleomorphs are known to be
members of the Tubeufiaceae (Kodsueb et al., 2006;Tsui &Berbee,
2006;Tsui, Sivichai, &Berbee, 2006;Boonmee et al., 2011,2014).
The taxonomy of species in these three genera has been tradi-
tionally morphological (Morgan, 1892;Linder, 1929,1931;Moore,
1953,1954,1955,1957). According to original generic circum-
scriptions, distinctions between Helicoma,Helicomyces and Heli-
cosporium were often vague due to similarities in coiling of their
conidia. Pirozynski (1972) suggested that the taxonomy of these
three genera could emphasize on the characters of conidiogenous
cells, conidial attachment position, presence of “conidiola”(sec-
ondary conidia), and presence of “sclerotes pedicelees”(stalked
sclerotia). To date, more than 200 species names have been
assigned into these three genera. Currently taxonomists of these
groups of fungi generally distinguish the three genera as follows: in
Helicomyces and Helicosporium, conidial filaments are relatively
thin in proportion to their length and hygroscopic (they uncoil in
water; Morgan, 1892). Conidiophores are well-developed in Heli-
cosporium (Goos, 1989) whereas in Helicomyces they are much
reduced or lacking (Goos, 1985). In Helicoma, conidia are non-
hygroscopic, and the conidial filaments are relatively thick in pro-
portion to their length (Goos, 1986).
To date, there are 40 names in Helicomyces (Index Fungorum,
2017), but many of these names have been synonymised,
excluded or transferred to more appropriate genera. Currently
there are 13 accepted names in Helicomyces (Boonmee et al., 2014;
Goos, 1985;Zhao, Liu, &Wu, 2007). During a survey of microfungi
occurring on plant litter submerged in a stream of Alishan area,
Chiayi County, Taiwan, we found several helicosporous fungi,
among them are Helicomyces torquatus L.C. Lane &Shearer (Lane &
Shearer, 1984) and an undescribed Helicomyces species, both
growing on decaying wood. The identities of these two Helicomyces
species were sought by seeking phylogenetic relationships among
similar taxa using available ITS sequences from Genbank, and
morphologically comparing with previously described species us-
ing a number of references (Linder, 1929,1931;Goos, 1985,1986,
1987,1989;Zhao et al., 2007; and; Cruz, Gusm~
ao, Le~
ao-Ferreira, &
Casta~
neda-Ruiz, 2009). In this paper, we describe and illustrate
H. torquatus as a new record for Taiwan, with new observation, and
H. geniculatus as a new species. Scanning electron micrographs of
H. torquatus are provided. A morphological comparison of accepted
Helicomyces species is given in Table 1.
*Corresponding author.
E-mail address: chkuo@mail.ncyu.edu.tw (C.-H. Kuo).
Contents lists available at ScienceDirect
Mycoscience
journal homepage: www.elsevier.com/locate/myc
https://doi.org/10.1016/j.myc.2018.04.002
1340-3540/©2018 Published by Elsevier B.V. on behalf of the Mycological Society of Japan.
Mycoscience 59 (2018) 433e440
2. Materials and methods
2.1. Sample collecting and mycological procedures
Sample collecting and mycological procedures were similar to
methodology described in Goh, Hyde, and Ho (1998). Plant litter
including wood were collected in plastic bags, and returned to the
laboratory where they were incubated at room temperature under
a humid condition in sterile plastic boxes. Materials were examined
periodically for the presence of fungal fruiting bodies and species
were identified. Single-spore isolation (Goh, 1999) of fungi were
made and grown in potato dextrose agar (PDA) slants and malt-
extract agar (MEA) plates at 20
C. The pure cultures were the
source of DNA for phylogenetic analysis.
2.2. Scanning electron microscopy
Fungal material was cut from natural substratum (decaying
wood submerged in freshwater) and then fixed by immersion in 2%
(W/V) aqueous osmium tetroxide (OsO
4
) for 12 h at 4
C in the dark.
Fixed material was washed in distilled water for 15 min to remove
excess osmium tetroxide, and then dehydrated in a 10% graded
ethanol series, 15-min steps from 10% to 90% ethanol. Material was
then washed in 95% ethanol followed by three 15-min changes of
absolute ethanol. Ethanol was replaced with acetone in 2:1, 1:2
(ethanol: acetone) steps followed by three changes of absolute
acetone (15 min each change). Dehydrated material was critical-
point dried, and then coated with platinum by a Hitachi E-1045
ion sputter coater at 15 mA and 7 Pa condition for 90 s, acquiring ca.
10 nm in thickness, and then examined in a Hitachi S-4700 Field
Emission Scanning Electron Microscope at 1 kV.
2.3. Fungal DNA extraction, rDNA barcoding polymerase chain
reaction (PCR) and DNA sequencing
Fungal isolates grown on PDA plates for 60 d were prepared for
DNA extraction. The protocol of DNA extraction was carried out
following Sambrook and Russell (2001). PCR amplification was
performed by using experimental sample cocktail, consisting of
2e8 ng DNA template, 0.4ng upstream primer and downstream
primer, PCR Master Mix II (5 ) (GeneMark Technology Co., Ltd.,
Taichung, Taiwan). Each of the PCR reactions in 25
m
L(1) total
volume contained 0.75 U of Taq DNA polymerase, 2 nM MgCl
2
,
250
m
M dNTPs, reaction buffer, and enzyme stabilizer. For the rDNA
barcoding, the primer set used to amplify the internal transcribed
spacer (ITS) region was ITS5 and ITS4. The amplification protocol
was: initial denaturation at 94
C for 2 min, followed by 35 cycles of
30 s at 94
C, 30 s at 50
C, and 30 s at 72
C, and concluded with a
final extension of 10 min at 72
C. The sequencing of the ITS se-
quences was achieved by using the same primer set used in PCR
Table 1
Morphological comparison accepted Helicomyces species
a
.
Species Setae Conidiophore
color
Conidiophore
size (
m
m)
Conidial
diam
(
m
m)
Conidial
filament
width
(
m
m)
Conidial
coiling
(no. of
times)
Sclerotia Conidiola Outstanding feature Reference
H. ambiguus
(Morgan) Linder
nil hyaline 130 2.5e3.5 30e40 5e73e5 nil nil Conidial base not swollen and not
eccentrically attached
Linder, 1929
H. bellus Morgan nil (N.A.) absent 15e25 1.5e3 2.5e3.5 nil nil Teeth on repent mycelium Morgan,
1892
H. colligatus nil hyaline 1845 2.5e4 (32)
5065
3.5e8 1.5e3 present present Robust conidiophores, conidia brittle;
conidial filament thick (8
m
m) but
tapering at both ends (3.5
m
m)
Moore, 1954
H. denticulatus G.Z.
Zhao, Xing Z. Liu &
W.P. Wu
nil hyaline 513 3e4.5 20e26 2e3.5 3.5e3.75 nil nil Short stout conidiophores with
denticles; conidia many coils
Zhao et al.,
2007
H. geniculatus present pale brown 234629e36 3e4.5 2.5e3.5 present nil Presence of mononematous setae
bearing geniculate conidiophores;
abundant stalked sclerotia
This paper
H. hyderbadensis P.
Rag. Rao &D. Rao
nil pale
yellowish
1028 3719e25 1.8e2.8 1.5e3.5 nil nil Small conidia; generally similar to
H. lilliputeus
Rao &Rao,
1964
H. lilliputeus R.T.
Moore
nil hyaline 2865 2611e28 1.5e2.5 1.5e3 nil nil Small conidia; distinctive branching
conidiophores
Moore, 1957
H. louisianensis Gooss nil pale brown 2575 2440e60 5e75e10 nil nil Conidia tightly coiled 5e7 times Goos, 1985
H. macrofilamentosus
Matsush.
nil pale brown 720 3435e55 5e12 2e3 nil nil Wide conidial filament (up to 12
m
m) Matsushima,
1983
H. roseus nil pale brown 1050 3.5e525e60 46(6.5) 2.25e3 present present Conidia borne on teeth from repent
mycelium or on short erect branches;
pinkish in mass
Linder, 1929
H. scandens present pale brown 7.540 3.5e515e35 1.5e32e3 nil nil Distinct synnema-like dark setae Morgan,
1892
H. tenuis nil hyaline micronematous 15e20 1.5e23e4 present present Small sized species producing slim
conidia from repent mycelium
Linder, 1929
H. torquatus
b
nil hyaline 1956 3.5e550e130 4.8e7.2 1.5e3 nil present Thick conidial filament filled with
abundant oil globules, tapering to a
swollen base, and becoming torque-
shaped in water.
Lane &
Shearer,
1984
H. torquatus
c
nil hyaline 2149 3.5e576e80 6.3e7.7 1.5e1.75 present present Conidial filament bearing multiple
conidiola; presence of stalked sclerotia
This paper
a
Helicomyces paludosa (P. Crouan &H. Crouan) Boonmee &K.D. Hyde is not included in this table because this taxon was proposed based on phylogenetic data of its
teleomorphic state only (Boonmee et al., 2014).
b
Data were based on the Panama collection (Lane &Shearer, 1984).
c
Data were based on the present Taiwanese collection.
C.-H. Kuo, T.-K. Goh / Mycoscience 59 (2018) 433e440434
Table 2
Taxa and sources of ITS sequences included in this investigation.
Fungal taxon Fungal strain Country of origin GenBank accession no.
Aquaphila albicans BCC 3543 Thailand DQ341096
Aquaphila albicans MFLUCC16-0010 Thailand KX454165
Cercospora apiicola CPC 11642 Greece DQ233341
Chlamydotubeufia helicospora MFLUCC16-0213 Thailand KX454169
Chlamydotubeufia khunkornensis MFLUCC10-0117 Thailand JN865201
Helicoma ambiens UBC F15000 Canada AY916451
Helicoma conicodentatum UBC F14998 Canada AY916450
Helicoma dennisii NBRC 30667 eAY916455
Helicoma guttulatum MFLUCC16-0022 Thailand KX454171
Helicoma miscanthi MFLUCC11-0375 Thailand KF301525
Helicoma morganii CBS 281.54 USA AY916468
Helicoma muelleri
b
CBS 964.69 Slovakia AY916453
Helicoma palmigenum NBRC 32663 Japan AY916480
Helicoma perelegans ATCC 22621 Japan AY916463
Helicoma rugosum UBC F13877 Canada AY916452
Helicoma siamense MFLUCC10-0120 Thailand JN865204
Helicoma vaccinii CBS 216.90 USA AY916486
Helicoma violaceum CBS222.58 Canada AY916469
Helicomyces bellus CBS 113542 Canada AY916475
Helicomyces geniculatus
a
BCRC FU30849 Taiwan LC335817
Helicomyces geniculatus
a
NCYU U2-1B Taiwan LC335816
Helicomyces lilliputeus NBRC 32664 Japan AY916483
Helicomyces paludosa AR 4206 Canada DQ341095
Helicomyces roseus
b
BCC 8808 Thailand AY916481
Helicomyces roseus
b
CBS 283.51 Switzerland AY916464
Helicomyces torquatus CBS 189.95 Cuba AY916472
Helicomyces torquatus
a
BCRC FU30844 Taiwan LC316605
Helicoon gigantisporum BCC 3550 Thailand AY916467
Helicosporium abuense CBS 101688 Puerto Rico AY916470
Helicosporium aureum NBRC 7098 eAY916478
Helicosporium griseum CBS 961.69 Czech Republic AY916474
Helicosporium griseum UAMH 1694 Canada AY916473
Helicosporium guianensis CBS 269.52 Netherlands AY916487
Helicosporium guianensis UAMH 1699 Canada AY916479
Helicosporium indicum CBS374.93 Australia AY916477
Helicosporium linderi NBRC 9207 Panama AY916454
Helicosporium lumbricoides JCM 9265 Japan AY916476
Helicosporium pallidum UBC F15001 Japan AY916462
Helicosporium pallidum CBS 962.69 Czech Republic AY916460
Helicosporium panachaeum CBS 257.59 Japan AY916471
Helicosporium phragmitis CBS 271.52 Netherlands AY916461
Helicosporium talbotii MUCL 33010 Malawi AY916465
Helicosporium vegetum
b
BCC 8125 Thailand AY916491
Helicosporium vegetum
b
BCC 3332 Thailand AY916490
Neoacanthostigma fusiforme MFLUCC11-0510 Thailand KF301529
Neoacanthostigma septoconstrictum MFLUCC15-1248 Thailand KX454176
Tubeufia asiana CT95 Canada DQ341097
a
New sequence generated in this study.
b
Type species of the genus.
C.-H. Kuo, T.-K. Goh / Mycoscience 59 (2018) 433e440 435
amplification. Sequencing was performed on an ABIPRISM 377DNA
sequencer at the Biotechnology Centre of National Chung Hsing
University.
2.4. Phylogenetic analysis
ITS region of our Helicomyces species was used for phylogenetic
analysis. Other sequences of similar taxa in the Tubeufiaceae were
retrieved from GenBank, and Cercospora apiicola M. Groenew.,
Crous &U. Braun was selected as the outgroup (Table 2). MUSCLE
was used for DNA alignment (Edgar, 2004). Poorly aligned positions
of DNA alignment were manually modified where necessary. All
positions containing gaps and missing data were eliminated.
Aligned sequences were analyzed using Mega7 (Kumar, Stecher, &
Tamura, 2016), and a phylogenetic tree inferred using the Neighbor-
Joining method (Saitou &Nei, 1987) was generated. The tree was
drawn to scale, with branch lengths in the same units as those of
the evolutionary distances used to infer the phylogenetic tree. The
confidence level of all trees was estimated with 1000 replicate
bootstrap analysis (Felsenstein, 1985). The evolutionary distances
were computed using the Maximum Composite Likelihood method
(Tamura, Nei, &Kumar, 2004) and are in the units of the number of
base substitutions per site.
3. Results
3.1. Taxonomy
Helicomyces geniculatus C.H. Kuo &Goh, sp. nov. Figs. 1 and 2.
MycoBank: MB 823426.
Etymology: geniculatus, referring to the distinct geniculation of
conidiophores.
Colonies on natural substratum effuse, white, arachnoic, cera-
ceous, coarsely flocculose, forming cottonlike mycelium layer.
Mycelium mostly superficial, partly immersed, composed of
branched, septate, subhyaline to pale brown, smooth hyphae.
Stalked sclerotia often present, yellowish brown to medium brown,
spherical, muriform, 14e23.5
m
m in diam. Setae arising from su-
perficial mycelium, branched, multiseptate, up to 120
m
m long,
4e6
m
m wide, uniform in width, subhyaline to pale brown, gradu-
ally becoming hyaline toward the rounded apex, sterile or bearing
conidiophores. Conidiophores macronematous, mononematous,
arising as lateral branches from creeping hyphae or as a single
lateral branch from the setae, short, unbranched, distinctly genic-
ulate due to sympodial proliferation of conidiogenus cell, subhya-
line to very pale brown, 58-septate, 23e50
m
m long, 4e6
m
m
wide. Conidiogenous cells polyblastic, sympodial. Conidia acroge-
nous, dry, holoblastic, attached eccentrically, seceding schizolyti-
cally from conidiogenous cells and leaving a refractive scar on the
conidiogenous loci, hyaline, hygroscopic, 29e36
m
m in diam.
Fig. 1. Helicomyces geniculatus (TNM F31100, holotype). A: Colonies on natural sub-
stratum. The erect setae are clearly visible. B: Group of conidia, setae, conidiophores,
and sclerotia. C: Branched hyphae, conidiophores, and sclerotia. D: Highlight of a
sclerotium. E: A conidiophore with geniculations, arising as lateral branch from the
shaft of branched setae, and producing conidia. F: A conidiophore with geniculations,
producing conidia. Bars: A 200
m
m; B¡F20
m
m.
Fig. 2. Helicomyces geniculatus (TNM F31100, holotype). A, B: Conidiophores bearing
conidia. C: A conidiophore showing distinct geniculations. D: A conidiophore arising as
a lateral branch from the seta. E¡K: Conidia. Bars:20
m
m.
C.-H. Kuo, T.-K. Goh / Mycoscience 59 (2018) 433e440436
Conidial filament 3e4(4.5)
m
m thick at the widest point, tapering
towards the ends, the proximal end slightly swollen and rounded,
indistinctly 10e18 ( 21)-septate, not constricted at the septa, coiled
(2.5)3e3.5 times. Conidiola absent. Teleomorph unknown.
HOLOTYPUS: TAIWAN, Chiayi County, Juchi Township, on
decaying wood submerged in a freshwater stream, 5 Feb 2017,
Chang-Hsin Kuo, NCYU-U2-1 (TNM F31100, deposited at the Na-
tional Museum of Natural Science, Taichung, Taiwan). Ex-type
culture BCRC FU30849, Bioresource Collection and Research
Centre (BCRC), Food Industry Research and Development Institute,
Hsinchu, Taiwan.
Notes: This species differs from other Helicomyces species in
having geniculate conidiophores arising from repent hyphae and
also borne as lateral branches from erect, mononematous, suhb-
hyaline to pale brown setae. Helicomyces scandens Morgan was the
first Helicomyces species that has been reported to have setae, but
they were clustered, bristle-like, stout and dark brown (Linder,
1929;Morgan, 1892;Zhao et al., 2007). Other than H. geniculatus,
stalked scletotia have also been reported in H. colligatus R. T. Moore,
H. roseus Link, and H. tenuis Speg. (Goos, 1985;Moore, 1954;Zhao
et al., 2007).
Helicomyces torquatus L. C. Lane &Shearer, Mycotaxon 19: 292
(1984) Figs. 3 and 4.
Colonies on natural substratum effuse, white, becoming brown
when aged, hairy, comprising of dense mass of large helical conidia.
Mycelium partly superficial and partly immersed, composed of
branched, septate, subhyaline to pale brown, smooth hyphae.
Stalked sclerotia often present, medium brown, spherical, muriform,
15e17(24)
m
m in diam. Setae absent. Conidiophores semi-
micronematous, mononematous, arising as lateral branches from
creeping hyphae, hyaline to very pale brown, 03-septate,
21e49
m
m long, 3.4e4.9
m
m wide. Conidia acrogenous, holoblastic,
produced singly, attached eccentrically, seceding schizolytically,
hyaline to pale grey, 76e80
m
m in diam. Conidial filament
354e475
m
m long, 6.3e7(7.7)
m
m thick at the widest point,
tapering towards the ends, the proximal end distinctly swollen and
rounded, closely 5861-septate, not constricted at the septa,
densely guttulate, coiled 1.5e1.75 times, hygroscopic and becoming
torque-shaped in water, bearing 1e4(6) conidiola. Conidiola
globose, 4.6e6
m
m diam, unicellular, rough-walled. Teleomorph
unknown.
Colonies in PDA culture blackish brown when aged, mycelia tend
to be immersed, producing large amount of spherical to cylindrical
chlamydospores (5e8
m
m diam) in branched chains, embedded in
the culture media.
Specimen examined: TAIWAN, Chiayi County, Juchi Township, on
decaying wood submerged in a freshwater stream, 5 Feb 2017,
Chang-Hsin Kuo, NCYU-K4-1, deposited at National Chiayi Univer-
sity, Chiayi County, Taiwan; single-spore culture FU30844, depos-
ited at Bioresource Collection and Research Centre (BCRC), Food
Industry Research and Development Institute, Hsinchu, Taiwan.
Notes: The morphological features of H. torquatus that we
collected from Chiayi County, Taiwan, agree very well with those
described in Lane and Shearer (1984) for a collection from Panama.
It is a new collection and record for Taiwan. The key features for
identification of this species include the large dimensions of its
conidia, the filament which is densely filled with oil globules, and
tapers to a swollen proximal end, the hygroscopic nature of the
conidia that often becoming torque-shaped in water, and the
presence of chained chlamydospores in culture. Our collection,
however, differs from the Panama collection in having abundant
stalked sclerotia, and each conidium bears several conidiola.
Known distribution: Hong Kong (Tsui, Hyde, &Hodgkiss, 2000),
Mainland China (Zhao et al., 2007), Panama (Lane &Shearer, 1984),
Taiwan.
3.2. Phylogenetic analysis
In this study, we generated three new rDNA barcodes, from two
isolates of H. geniculatus a(Genbank accession numbers LC335817
and LC335816) and one isolate of H. torquatus (Genbank accession
number LC316605), which were 1022 bp,1031 bp, and 619 bp long,
respectively. These sequences were used in the present phyloge-
netic study, comparing them with those of similar taxa from the
Tubeufiaceae.
The phylogenetic tree inferred from ITS sequences (Fig. 5;
TreeBase TB2:S21876) showed phylogenetic relationships among
47 taxa of fungi, including H. geniculatus,H. torquatus, and other
representative taxa from the Tubeufiaceae. After alignment, all
positions containing gaps and missing data were eliminated. There
were a total of 325 positions in the final dataset, which were
informative characters. Molecular data revealed several small
clusters (AK) of taxa, with species of Aquaphila,Chlamydotubeufia,
and Neoacanthostigma basically in their distinct clades, but species
of Helicoma,Helicomyces and Helicosporium were intermixed
Fig. 3. Helicomyces torquatus (NCYU K4-1). A: Colonies on natural substratum. B¡E:
Stalked sclerotia. F¡I: Conidiophores. J¡P: Conidia which bear conidiola; Jand Kwere
mounted in lactophenol cotton blue. Q¡S: Highlights of the conidiola on conidial fil-
aments. T: Colony from single-spore culture on PDA. U: Chains of chlamydospores
found in culture. Bars:A200
m
m; B¡I10
m
m; J¡P20
m
m; Q¡S10
m
m; T10 mm; U
20 mm.
C.-H. Kuo, T.-K. Goh / Mycoscience 59 (2018) 433e440 437
among the clades. Majority of Helicomyces species, including an
isolate (BCC 8808) of H. roseus (the type species of the genus), two
isolates of H.geniculatus,H. lilliputeus,H. torquatus (Taiwanese
collection), and Helicoma perelegans formed one distinct clade
(Clade A) with a bootstrap support of 72%. The other isolate of
H. roseus (CBS 283.51), however, clustered with two species of
Helicosporium,Helicoma palmigenum, and Helicomyces paludosa in
Clade C. Helicoma muelleri (the type species of the genus) clustered
with majority of Helicoma species in Clade G, whereas other Heli-
coma species were interspersed amongst other clades. The two
isolates of Helicosporium guianense did not cluster together: one
(CBS 269.52) clustered with the two isolates of Helicosporium
vegetum (the type species of the genus) in Clade K, but the other
(UAMH 1699) with Helicosporium aureum in Clade H. Other species
of Helicosporium were also interspersed amongst various small
clusters of taxa.
4. Discussion
According to our phylogenetic tree (Fig. 5), species of Helicoma,
Helicomyces, and Helicosporium were interspersed among various
clusters of taxa. This result concurs with many previous phyloge-
netic studies that these helicosporous fungi, although clearly
belonging to the Tubeufiaceae, they are not monophyletic at
generic separation (Kodsueb et al., 2006;Tsui &Berbee, 2006;Tsui
et al., 2006;Boonmee et al., 2011,2014). Certainly there are still
rooms for further investigations of these hyphomycetes, however, it
is out of the scope of the present paper to discuss generic level
systematics or to resolve the phylogenetic relationships of these
three genera.
The two isolates of Helicomyces geniculatus (BCRC FU30849 and
NCYU U2-1B) in our phylogenetic study distinctly clustered with
the majority of Helicomyces species in Clade A (Fig. 5), including one
isolate of H. roseus (type species of the genus). The morphological
features in H. geniculatus also agree well with those of the type
species and other typical Helicomyces species. Based on the present
phylogenetic position inferred by the rDNA barcode analysis, and
also with careful morphological comparison with similar species of
Helicomyces, we are therefore confident to describe H. geniculatus
as a new species.
In our phylogenetic study, the Taiwanese collection of
H. torquatus (BCRC FU30844; Genbank accession number:
LC316605) clustered well in a clade comprising H. roseus (BCC
8808) and several other Helicomyces species. The morphological
features of our collection also agreed well with those described and
illustrated in Lane and Shearer (1984). We are therefore confident
in our identification of H. torquatus. The only available ITS sequence
of H. torquatus deposited in Genbank (AY916472) was from a Cuban
isolate (CBS 189.95). Nevertheless, it did not cluster with the
Taiwanese collection in Clade A, instead clustered with a major
group comprising mainly of Helicosporium species (Clades D and F),
and two Helicoma species (Clade E). Without examination of
“H. torquatus”from Cuba, it is hard to judge if the Cuban collection
may be misidentified and belonged to Helicosporium. Most char-
acters in defining these helicosporous fungi are rather plastic,
convergent evolution could be one of the reasons for the discrep-
ancies in the placement of the fungi with identical names.
While the two Helicomyces torquatus isolates (Taiwanese and
Cuban) did not cluster together in our phylogenetic tree, the two
isolates of the type species, Helicomyces roseus, one from Thailand
(BCC 8088) and the other from Switzerland (CBS 283.51), also did
not cluster together. Similarly, the two Helicosporium guianense
isolates (UAMH 1699 and CBS 269.52) were in different clades
(Clades H and K). Tsui et al. (2006) in their phylogenetic studies of
helicosporous fungi also have discussed the same result. We did not
attempt to resolve the problem, as it is out of the scope of the
Fig. 4. Helicomyces torquatus (NCYU K4-1), scanning electron micrographs. A: Group of conidia on natural substratum. B: Conidia with conidiola (arrowed). C: Higher magnification
of the conidiola. D: Conidiophores arising as short erect lateral branches from repent hyphae, with conidia attached at the tip. Bars:A50
m
m; B20
m
m; C5
m
m; D20
m
m.
C.-H. Kuo, T.-K. Goh / Mycoscience 59 (2018) 433e440438
Fig. 5. Phylogenetic tree (TreeBase TB2:S21876) inferred from aligned ITS sequences of 47 fungi using neighbor-joining method, showing relationships of Helicomyces geniculatus,
H. torquatus, and representative taxa from the Tubeufiaceae, with Cercospora apiicola being the outgroup. The optimal tree with the sum of branch length ¼1.42404776 is shown.
Support values from bootstrap test (1000 replicates) greater than 50% are shown near each node. The tree is drawn to scale, with branch lengths in the same units as those of the
evolutionary distances used to infer the phylogenetic tree.
C.-H. Kuo, T.-K. Goh / Mycoscience 59 (2018) 433e440 439
present study.
Acknowledgments
We would like to thank Mr. Jie-Hao Ou for his help in DNA
isolation and phylogenetic analysis of our fungi. We are also
grateful to Dr. Sung-Yuan Hsieh of Bioresource Collection and
Research Centre at Hsinchu for his help in scanning electron
microscopy.
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